This invention relates to the chemical encryption of the structure of compounds formed in situ on solid supports by the use of specific amine tags which, after compound synthesis, can be deencrypted to provide the structure of the compound found on the support.
The solid supports of this invention find particular utility in preparing encoded synthetic libraries of compounds on the support for facilitating screening of these compounds for biological activity.
The following publications and patent applications are cited in this application as superscript numbers:
1 M. A. Gallop, R. W. Barrett, W. J. Dower, S. P. A. Fodor and E. M. Gordon, J. Med. Chem., 37:1233 (1994)
2 E. M. Gordon, R. W. Barrett, W. J. Dower, S. P. A. Fodor and M. A. Gallop, J. Med. Chem., 37:1385 (1994)
3 A. Furka, F. Sebestyen, M. Asgedom and G. Dibo, Int. J. Peptide Protein Res., 37:487 (1991)
4 W. J. Dower, R. W. Barrett and M. A. Gallop, International Patent Application Publication No. WO 93/06121 (1993)
5 S. Brenner and R. A. Lerner, Proc. Natl. Acad. Sci., USA, 89:5181 (1992)
6 J. M. Kerr, S. C. Banville and R. N. Zuckermann, J. Am. Chem. Soc., 115:2529 (1993).
7 V. Nikolaiev, A. Stierandova, V. Krchnak, B. Seligmann, K. S. Lam, S. E. Salmon and M. Lebl, Pept. Res., 6:161 (1993)
8 M. C. Needels, D. G. Jones, E. M. Tate, G. L. Heinkel, L. M. Kochersperger, W. J. Dower, R. W. Barrett and M. A. Gallop, Proc. Natl. Acad. Sci., USA, 90: 10700 (1993)
9 M. H. J. Ohlmeyer, R. N. Swanson, L. W. Dillard, J. C. Reader, G. Asouline, R. Kobayashi, M. Wigler and W. C. Still, Proc. Natl. Acad. Sci. USA, 90:10922 (1993)
10 C. P. Holmes, International Patent Application Serial No. PCT/US95/07988 for xe2x80x9cMethods for the Solid Phase Synthesis of Thiazolidinones, Metathiazanones, and Derivatives Thereofxe2x80x9d, filed Jun. 23, 1995
11 Lebl, et al., Peptide Science, February 1995
12 Campbell, et al., International Patent Application Serial No. PCT/US95/07964, for xe2x80x9cMethods for the Synthesis of Diketopiperazinesxe2x80x9d, filed Jun. 23, 1995
13 Gallop, et al., International Patent Application Serial No. PCT/US95/07878, for xe2x80x9cMethods for Synthesizing Diverse Collections of Pyrrolidine Compoundsxe2x80x9d, filed Jun. 22, 1995
14 Gallop, et al., U.S. patent application Ser. No. 08/264,136 for: xe2x80x9cMethods for Synthesizing Diverse Collections of xcex2-Lactam Compoundsxe2x80x9d, filed Nov. 21, 1995
15 Farina, et al., J. Med. Chem., 3:877 (1991)
16 Stills, et al., International Patent Application Serial No. PCT/US93/09345 for xe2x80x9cComplex Combinatorial Chemical Libraries Encoded with Tagsxe2x80x9d, filed Oct. 1, 1993
All of the above publications and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Synthetic chemical libraries produced by combinatorial synthesis have rapidly become important tools for pharmaceutical lead discovery and compound optimization.1,2 Typically, combinatorial synthesis is conducted via a multi-step synthesis to provide a library of target compounds. Each step in this synthesis involves a chemical modification of the then existing molecule formed from the previous step wherein one can vary the choice of reagents and/or reaction conditions to provide for a variety of different target compounds. For example, such steps could include the use of different building blocks to form different compounds, the use of different inorganic or organic reagents which alter where the building blocks are added or the stereochemistry of the addition, etc.
Many of the combinatorial approaches devised to prepare such libraries rely on solid-phase synthetic techniques and exploit the efficient xe2x80x9csplit/poolxe2x80x9d method to assemble all possible combinations of a set of chemical building blocks.3 The xe2x80x9csplit/poolxe2x80x9d method employs a pool of solid supports which contains or can be derivatized to contain reactive moieties for forming the molecules of interest tethered to the solid support. This pool is initially split and each split pool is then subjected to a first reaction which reaction results in different modifications to each of the pools. After reaction, the pools of solid supports are combined and the pooled supports are then again split. Each split pool is subjected to a second reaction which is different for each of the pools. The process is continued until a library of target compounds are formed on the solid supports.
The reactions employed at each stage of this synthesis can include the addition of different building blocks to the solid support, the use of different reagents and/or reaction conditions to differentially alter the existing chemical entity on the solid support, etc. Also combinations of different building blocks with different reagents and/or reaction conditions can also be employed.
The xe2x80x9csplit/poolxe2x80x9d protocol is particularly well-suited to the generation of large libraries, and the synthetic target compounds may be screened for interaction with macromolecular receptors either in binding assays where the compounds remain tethered to their synthetic supports, or in soluble assays after cleavage of the compounds from the resin. Elucidation of the chemical structure of biologically active library members has represented a major challenge because the quantity of material available for chemical analysis from a complex library is frequently minuscule.
A general solution to this structure eludication problem has been proposed that exploits a set of surrogate analytes, or identifier tags, which can be detected with either greater ease and/or sensitivity than the chemical entities which they represent.4 Through their concurrent appendage to the synthesis supports, these tags provide an unambiguous record of chemical reaction history and/or chronology of monomer (building block). additions to each support in the library. This method, which has become known as encoded combinatorial synthesis5, has broad scope and utility, and conceptually may be applied to the construction of any collection of compounds that can be produced through a multi-step scheme of synthesis on solid supports.
Two conceptually different approaches to encoding a combinatorial synthesis have been described. In the first mode, the sequence of monomer addition steps is recorded by the parallel and alternating assembly of a polymeric molecule that is itself amenable to chemical sequence analysis. Here the structure of any combinatorial product is reflected by the sequence of a single cognate identifier tag on the solid support. Both peptides6,7 and oligonucleotides8 have been sucessfuly used in this manner to encode the synthesis of polyamide combinatorial libraries, the tags being analyzed by, for example, Edman or dideoxy Sanger sequencing respectively.
In the second encoding method, a set of readily identifiable unique markers employed for each reaction step is attached to the solid support to identify which reaction was visited in which step of the target compound synthesis on that particular support.4,9 The set of identifiable markers is employed in binary code format with each set identifying a different monomer and/or reaction conditions. For example, each marker can be represented in binary code format by either a xe2x80x9c0xe2x80x9d for its absence or a xe2x80x9c1xe2x80x9d for its presence. Accordingly, if 3 different identifiable markers are employed in a first set, this set contains 7 unique binary code combinations (the binary code represented by 000 is not used). Specifically, the binary codes for these combinations employing markers A, B and C would read as follows:
In this example, the use of 7 different building blocks for a given step in the xe2x80x9csplit/poolxe2x80x9d synthesis could be encoded by the above set of 3 markers using the 7 different combination of these markers. Each different combination of markers provides a xe2x80x9ctagxe2x80x9d for a particular building block used in the synthesis.
As is apparent, increasing the number of markers from 3 to 10 in the set would increase the number of unique binary combinations for that set to over 1000. The large number of combinations is necessary since an n-step synthesis using m building blocks at each step requires that total of mxc3x97n monomer-specific steps be separately identified.
Removal of all markers from the solid support and identification of their presence or absence identifies the particular tag defined by that marker combination and, accordingly, the relevant information concerning the monomer addition. In this embodiment, the compounds employed as identifiable markers in the encryption set must be distinguishable from each other in order to be capable of accurate deencryption and the compounds employed in a first set must be different from the compounds employed in a second set in order to maintain strict correlation between the encryption/deencryption information.
Central to this approach is the understanding that the binary code provides for a significantly larger pool of possible tag combinations than a monomeric code. For example, if the synthetic scheme encodes for three different reaction steps, the use of 30 unique compounds in three sets of 10 identifiable markers each would encode for at least 10003 or 109 possible combinations. In this regard, Still, et al.9 have described how a series of 18 chromatographically resolvable different halocarbon derivatives can be appended to a synthesis resin in a binary coding strategy, and subsequently liberated for analysis by electron capture gas chromatography to code for a library of 117,649 peptides.
Essential requirements with any scheme of encoded combinatorial synthesis on solid supports is that the chemistries employed in compound synthesis and tag addition steps be mutually compatible, that the tags remain on the support during each step of the synthesis, and that the tag uniquely identify a particular reaction step. An ideal tagging medium would, therefore, be one which reacts robustly with functional groups found on or attached to the resin so as to be incorporated thereon in high yield, which tagging reactions are completely chemically inert to the reaction intermediates and products employed in compound synthesis and, which tags are amenable to rapid and straightforward analysis at trace levels.
In this regard, considerable success has been achieved in using oligonucleotides as an enzymatically amplifiable coding moiety, and it has been demonstrated that the DNA tagging strategy is sufficiently versatile and robust to permit the automated synthesis of encoded libraries of glycopeptides, polycarbamates and thiazolidinones10, in addition to polyamides. Nevertheless, since oligonucleotides do possess sites of chemical lability and the chemistry for oligonucleotide synthesis is not always compatible with compound synthesis especially of small molecules1, there is a continuing need in the art to develop encoding strategies complementary to the synthesis of small organic molecule libraries.
This invention is directed to chemical entities useful as tags on solid supports to encode a target compound synthesized on the support. These tags are reactive with functional groups on or attached to the support and provide for an unambiguous record of the reaction history including the chronology of monomer (building block) additions during target compound synthesis on the support. Moreover, the tags are inert to the organic synthesis scheme employed to prepare the target compound on the support surface and can be deencrypted at an appropriate point in time in order to determine the structure of the target compound formed on the support.
More specifically, this invention is directed to the use of chemical tags and solids supports containing these tags covalently attached thereto. These tags are an amine or mixture of amine tags selected from a plurality of amines of formula Ia: 
wherein R and Rxe2x80x2 are independently hydrocarbyl groups of from 1 to 30 carbon atoms which define a unique amine tag used to identify a reaction conducted in target compound synthesis and/or the point in time where said reaction was conducted; R4 and R5 are either hydrogen or are joined to form a piperidine ring; Pg is selected from hydrogen, an amine tag of formula Ia above bound to the amino nitrogen through the carbonyl functionality of formula Ia and a compatible protecting group provided that the compatible protecting group is orthogonal to any and all protecting groups employed in target compound synthesis, and Pgxe2x80x2 is selected from xe2x80x94OH, an amine tag of formula Ia above bound to an amino nitrogen through the carbonyl functionality of formula Ia and xe2x80x94OPgxe2x80x3 where Pgxe2x80x3 is a compatible protecting group which is orthogonal to any and all protecting groups employed in target compound synthesis.
The amine compounds of formula Ia above are covalently attached to the solid support either directly or through a suitable linker arm through either the amine or carboxyl functionality.
In view of the above, in one of its composition aspects, this invention is directed to a solid support comprising multiple copies of a single target compound covalently attached thereto wherein the target compound is prepared in situ on the support by sequentially conducting at least two reactions wherein each of the reactions employed to prepare said target compound and/or the step in the chemical synthesis where said reaction is conducted is encoded by a tag covalently coupled to the support, immediately before or after each reaction, wherein said tag is an amine or mixture of amines selected from a plurality of amines of formula I 
wherein R and Rxe2x80x2 are independently hydrocarbyl groups of from 1 to 30 carbon atoms which define a unique amine tag used to identify a reaction conducted in target compound synthesis and/or the point in time where said reaction was conducted; R4 and R5 are either hydrogen or are joined to form a piperidine ring; and Pg is selected from hydrogen, an amine tag of formula I above bound to the amino nitrogen through the carbonyl functionality of formula I and a compatible protecting group provided that the compatible protecting group is orthogonal to any and all protecting groups employed in compound synthesis,
provided that each tag combination used to encode a first reaction and/or the point in time where said reaction is conducted is different from and distinguishable over all other tag combinations used to encode the remaining reactions and/or points in time where said reactions are conducted so as provide a binary or higher coding system wherein the code uniquely identifies the compound resulting from the reactions.
The amine compounds set forth above define a pool of compounds useful for forming sets of tags each set of which can encrypt for each variant employed in a single reaction involved in the synthesis of target compounds and/or the point in time where said reaction is conducted. In order to unequivocally resolve each encrypted tag, the compounds employed in each tag set must be different from and distinguishable over the compounds employed in the other sets.
In one embodiment, the tag is different than the other tags based on chemical identity. In this embodiment, the tag for a first reaction is selected to provide a unique combination from a plurality of individual amines compounds included within the selected set of these compounds. For example, if the first reaction merely employs different building blocks, a set of three compounds (markers) A, B and C provides for 7 unique tag combinations in binary code format (other than 000) for identifying the 7 different building blocks employed in this first reaction. Each unique tag combination unequivocally encrypts for each building block.
Chemically different tags include those having different chemical structures including both positional and stereochemical isomers as well as those having the same structure but which have, for example, radioisotopes at different locations in the molecular structure. The different radioisotopes can be distinguished from each other based on, for example, proton or 13C nuclear magnetic resonance.
Ternary codes can be based on, for example, chemical identity and the presence of a compound at two different concentrations. In this case, the three states defining the ternary code would be the presence of the compound at two different and distinguishable concentrations or its absence. The advantage of higher order codes over a binary code is that fewer compounds are required to encode the same quantity of information about the synthesis or, using the same number of compounds, more information can be encoded.
Each amine compound is distinguishable over the remaining amine compounds based on some physical or chemical property. The distinguishing feature can be found in the compound itself or a derivative thereof and includes, by way of example, retention times on HPLC, nuclear magnetic spectrum profiles (either 1H or 13C), mass spectroscopy, etc.
Preferably, when amines of formula I form the tag, the tags are constructed to form a polymeric amide on the solid support which polymeric amide has the formula 
wherein the polymeric amide is covalently attached to the solid support either directly or through a linker arm and further wherein each R and Rxe2x80x2 define a unique amine tag used to identify a reaction conducted in target compound synthesis and/or the point in time where said reaction was conducted, R4 and R5 are either hydrogen or are joined to form a piperidine ring, Pg is a compatible protecting group, and n represents the number of reactions encoding for the target compound synthesis provided that each amine tag combination employed to encode a single reaction is different from and distinguishable over all other amine tag combinations used to encode other variations used in that reaction and still further provided that the amines used to encode a first reaction are different from the amines used to encode the other encrypted reactions so as provide a binary or higher coding system wherein the code uniquely identifies the target compound resulting from monomer coupling.
These polymeric amides are formed from the monomeric amines of formula I merely by converting the Pg group to hydrogen and then reacting the resulting amine (xe2x80x94NH) with the carboxyl group (or activated carboxyl group) of a second monomeric amine compound of the formula RRxe2x80x2NC(O)CH2NPgCH2COOH wherein R, Rxe2x80x2 and Pg are as defined above. For n encryptions, this process is repeated n times. At this point, the terminal amine of the polymeric amide contains a xe2x80x94NPg group which can be readily deblocked to provide the xe2x80x94NH group.
The use of such polymeric amides permits chemical deencryption simply by breaking the amide bonds to form a plurality of n sets of different amines which are then analyzed to determine the presence or absence of specific amines.
In view of the above, in another of its composition aspects, this invention is directed to a solid support comprising multiple copies of a single target compound covalently attached thereto wherein the target compound is prepared in situ on the support by sequentially conducting at least two reactions wherein each of the reactions employed to prepare said target compound and/or the step in the chemical synthesis where said reaction is conducted is encoded by a monomeric unit of a polymeric amide of the formula 
wherein the polymeric amide is covalently attached to the solid support and further wherein R and Rxe2x80x2 of each monomeric unit define a unique amine tag used to identify a reaction conducted and/or the point in time when said reaction was conducted in the target compound synthesis, Pg is a compatible protecting group, R4 and R5 are either hydrogen or are joined to form a piperidine ring, and n represents the number of reactions encoding for the target compound synthesis provided that each amine compound employed in tags to encode a single reaction and/or point in time when said reaction is conducted is different from and distinguishable over all other amine compounds employed in tags used with the other encryptions so as provide a binary or higher coding system wherein the code uniquely identifies the target compound resulting from the reactions conducted on the solid support.
In one of its method aspects, this invention is directed to a method for tagging a solid support having multiple copies of a single target compound covalently attached thereto in order to determine the structure of the target compound attached to the support wherein the compound is prepared in situ on the support by sequentially conducting n reactions on said support wherein n is an integer greater than 1 which method comprises:
a) conducting a first reaction on the solid support wherein the first reaction and/or the step in the chemical synthesis where said reaction is conducted is encoded by a tag coupled to the support, immediately before or after coupling of each monomer, wherein said tag is an amine or mixture of amines selected from a plurality of amines of formula I 
wherein R and Rxe2x80x2 are independently hydrocarbyl groups of from 1 to 30 carbon atoms which define a unique amine tag used to identify the reaction 4F conducted in target compound synthesis and/or the point in time where said reaction is conducted; R4 and R5 are either hydrogen or are joined to form a piperidine ring; and Pg is selected from hydrogen, an amine tag of formula I above bound to the amino nitrogen through the carbonyl functionality of formula I, a compatible protecting group provided that the compatible protecting group is orthogonal to any and all protecting groups employed in compound synthesis, and hydrogen provided that one of the Pg groups is hydrogen
provided that each tag combination employed to encode a single reaction is different from and distinguishable over all other tag combinations used to encode other variations used in that reaction and still further provided that the amine compounds used to encode a first reaction are different from the amine compounds used to encode the other encrypted reactions so as provide a binary or higher coding system; and
b) repeating procedure a) above until all n steps for target compound synthesis on the support are completed.
The tags described above are particularly useful for encoding target compounds prepared in libraries via combinatorial chemistry wherein each support contains multiple copies of a single target compound. Accordingly, in this embodiment, this invention is directed to a library of target compounds bound to a solid support wherein each support comprises multiple copies of a single target compound covalently attached thereto wherein each target compound in the library is prepared in situ on the support by sequentially conducting at least two reactions wherein at least one of the reactions conducted to prepare a first target compound is different from the reactions conducted to prepare the remaining target compounds and further wherein each of the reactions conducted to prepare each target compound and/or the step in the chemical synthesis where said reaction is conducted is encoded by a tag coupled to the support having said compound bound thereto, immediately before or after coupling of each monomer, wherein said tag is an amine or mixture of amines selected from a plurality of amines of formula I 
wherein R and Rxe2x80x2 are independently hydrocarbyl groups of from 1 to 30 carbon atoms which define a unique amine tag used to identify the reaction conducted in target compound synthesis and/or the point in time where said reaction is conducted; R4 and R5 are either hydrogen or are joined to form a piperidine ring; and Pg is selected from hydrogen, an amine tag of formula I above bound to the amino nitrogen through the carbonyl functionality of formula I and a compatible protecting group provided that the compatible protecting group is orthogonal to any and all protecting groups employed in compound synthesis
provided further that each tag combination employed to encode a single reaction is different from and distinguishable over all other tag combinations used to encode other variations used in that reaction and still further provided that the amine compounds used to encode a first reaction are different from the amine compounds used to encode the other encrypted reactions so as provide a binary or higher coding system.